Evaluation of a commercial multiplex immunoassay for assessing uterine inflammation in mares susceptible to persistent breeding-induced endometritis.

Persistent breeding-induced endometritis (PBIE) is characterized by a prolonged and dysregulated uterine inflammatory response following breeding. Although inflammatory cytokines are central to the post-breeding immune response, their assessment in the equine uterus has historically relied largely on gene expression analyses. Multiplex immunoassays provide an opportunity to simultaneously quantify multiple cytokine proteins within uterine secretions; however, the application of equine-specific multiplex immunoassays utilizing the Luminex platform to low-volume uterine lavage (LVL) fluid remains poorly characterized as a method for assessing post-breeding uterine inflammation. Therefore, the objective of this study was to evaluate whether cytokine concentrations quantified using an equine-specific Luminex multiplex immunoassay reflect established clinical and cytologic measures of uterine inflammation following a standardized breeding challenge in mares susceptible to PBIE. Six mares previously classified as susceptible to PBIE underwent intrauterine insemination with freeze-killed spermatozoa during three estrous cycles (n = 18 breeding challenges). Uterine inflammation was assessed post-insemination by transrectal ultrasonography, endometrial cytology obtained by cytobrush and low-volume lavage (LVL), and quantification of luminal and systemic cytokines using an equine-specific multiplex immunoassay. Among the cytokines evaluated, CXCL-8 demonstrated the strongest association with neutrophil infiltration, whereas IL-1β exhibited only a weak relationship and IL-6 and TNF were not associated with inflammatory status. Luminal CXCL-8 concentrations were positively associated with neutrophil counts recovered by LVL (R² = 0.66, p < 0.001) and cytobrush cytology (R² = 0.37, p < 0.001), while a weaker association was observed with uterine fluid accumulation (R² = 0.15, p = 0.02). No relationship was identified between luminal and systemic CXCL-8 concentrations (p = 0.70), nor between systemic CXCL-8 and cytologic indicators of inflammation. These findings demonstrate that CXCL-8 concentrations quantified in LVL fluid using an equine-specific Luminex multiplex immunoassay closely reflect neutrophil-mediated uterine inflammation following breeding challenge.